Malaria is one of the most serious protozoal infections in man. According to estimation made in the 90's, about 300 to 500 million people develop clinical infection and one million die of severe infection every year. India is also among the countries to have endemic regions of the disease. It is, therefore, of prime concern and requirement to have therapeutically safe agents for multiple use, especially those that block transmission of malaria through the individuals visiting endemic regions. A recent report of resurgence of malaria after a long gap of 40 years from Italy through transmission, highlights our concern [The Lancet, 350, 717 (1997)].
Malaria is caused by infection with any one of the four species of Plasmodia. The life cycle of Plasmodia is complex and comprises a sexual phase (called sporogyny) in the mosquito (a vector) and an asexual division (called schizogyny) in humans. The life cycle starts after injection of sporozoites by the bite of an infected female anopheline mosquito. Sporozoites then rapidly enter into liver parenchymal cells where they undergo exoerythrocytic schizogony forming exoerythrocytic stage of tissue schizonts which mature and release thousands of merozoites in the bloodstream upon the rupture of infected cell. Some of these merozoites enter erythrocytes where they transform into trophozoites and schizonts. The mature schizonts rupture and release merozoites into the circulation, which can infect other erythrocytes. This is termed as asexual schizogony (erythrocytic cycle) and it is this periodic release of merozoites which is responsible for characteristic periodicity of the fever in malaria. After several erythrocytic cycles, some erythrocytic forms differentiate into sexual forms called gametocytes. In P. vivar. and P. ovale infections, some of the sporozoites after entering the liver cells are known to remain dormant and form the latent tissue stage called hypnozoites. These hypnozoites upon activation develop secondary tissue schizonts, which are responsible for the recurrence of malaria called relapsing malaria. The 8-aminoquinoline antimalarial drugs of which primaquine (PQ) is of exceptional importance, have been demonstrated to possess activity against several life cycle stages of the parasite. These agents are active against the primary tissue schizonts, thus functioning as causal-prophylactic agents, against the secondary exoerythrocytic forms and curing relapsing forms of malaria. The transmission of malaria as discussed earlier, is through the injection of sporozoites by the bite of mosquitoes. These sporozoites develop in the mosquito feeding on an individual carrying mature gametocytes. The male and female gametocytes upon ingestion by a female anopheline mosquito fertilize and transform into zygote and ookinete stages. The ookinetes pierce through the epithelium of the midgut where it rounds up into the oocyst. A single oocyst contains as many as 10000 sporozoites. Primaquine has no sporontocidal activity when provided directly to the insects but has strong gametocytocidal activity and even stops transmission of resistant isolates when mosquitoes are fed on infected blood from primaquine treated animals. Thus, primaquine is also a strong transmission blocking agent. However, primaquine even being associated with radical curative and gametocytocidal activities is not in use as a prophylactic agent.
The practical problems associated with use of 8-aminoquinolines are mainly related to their toxicity because of prolonged use in radical treatment required due to fast metabolism of the drug. Primaquine is known to induce hemolytic lesions in patients suffering from a deficiency in glucose-6-phosphate-dehydrogenase (G-6PD), a genetic condition common among inhabitants in regions where malaria is endemic. Anemia is a common complication of hemolysis. Primaquine produces metabolites like o-quinone and p-quinomine functionalities, which because of their oxidative nature, oxidise unsaturated fatty acid of erythrocytes causing red blood cell (RBC) lysis. The reduced glutathione (GSH) controls the level of oxidative metabolites and the level of GSH is maintained through NADPH controlled GSSG reduction. NADPH is regulated by G-6PD and hence G-6PD deficient patients are more liable to RBC lysis. Primaquine is the only antimalarial drug, which inhibits the development of the parasite by interfering at several stages of the parasitic life cycle and therefore an ideal molecule for structural modification to provide a molecule with radical curative and gametocytocidal activities with low toxicity The study of the fate of primaquine, its metabolites and toxic manifestation in relation with metabolites will therefore, guide the direction of changes in the new molecule. A brief discussion of primaquine metabolism is given here.
Following oral administration of labelled primaquine it was found that 45% of the radioactivity was found in liver tissue, and 22% in the lung, adrenal, spleen, kidney, heart, blood and pancreas while 25% reached in to the plasma. Thus, primaquine is fairly well absorbed and only a small portion actually reaches the plasma.
Primaquine metabolism occurs at two sites of the molecule: one in the aromatic region at 5- and 6-positions and the other at 8-N aminoalkyl side chain. The first metabolic pathway leads to the formation of 5-hydroxyprimaquine (5-HPQ, 3), 5-hydroxy-demthyl primaquine (5-HDPQ) of the formula (4).

The second pathway originally observed to occur in the microorganisms, affects the 8-N-aminoalkyl chain and results in the formation of N-acetylprimaquine and desamino carboxylic acid of the Formula (12).

The carboxylic acid derivative is the major metabolite of primaquine in the human plasma.
Strother et al identified identified metabolites from the urine of primaquine treated dogs as 5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino) quinoline of the Formula (3), desmethyl-6-hydroxy-8-(4-amino-1-methylbutylamino) quinoline of the Formula (9) and 5,6-dihydroxy-6-methoxy-8-(4-amino-1-methylbutylamino) quinoline of the Formula (4) shown below: [A. Strother, et al, ‘Metabolism of *-amonoquinoline antimalarial agents’. Bulletin of the World Health organisation, 59, 413–425 (1981)].
Among N-dealkylated derivatives of primaquine metabolites were identified as 6-methoxy-8-aminoquinoline of formula (10) [J. D. Baty et al ‘The identification of 6-methoxy-8-aminoquinoline as a metabolite of primaquine in Man’. Biomedical Mass Spectrometry, 2, 304–306 (1975)] and 8-(3-carboxy-1-methylpropylamino)-6-methoxy quinoline of formula (12) shown below. [J. K. Baker, et al ‘HPLC analysis of the metabolism of primaquine and the identification of a New Mammalian Metabolite’ Journal of Chromatography, 230, 69–77 (1982)].
A blue colour metabolite derived from 5-hydroxy-desmethylprimaquine was identified as tricyclic quinomine of formula (8) shown below [A. Strother et al ‘Metabolism of Primaquine by various Animal species’ in Primaquine: Pharmacokinetics, Metabolism, Toxicity and Activity, pp 27–48 (1984), John Wiley & Sons].
Therapeutic Activity of Primaquine and its Metabolites
Primaquine has blood schizontocidal activities whereas its desmethyl derivative has little. Two 5-OH derivatives of the formula of (3) and (4) shown above are highly active. The quinolines that lack the side chain of 8-position but have merely amino substituents shown in the formula (10) above and formula (11) below have no significant activity.

In marked contrast is the observation that the dealkylated derivatives of the formulae 10 and 11 retain their tissue schizontoidal effect. They are two to three times more active than primaquine.
The direct sporontocidal activity of PQ and of these putative metabolites is poor against the oocysts development when mosquitoes are fed on treated animals that supply the gametocytes. Primaquine is quite inactive as sporontocide when given directly to the insect, but is a very potent gametocytocidal agent.
The 5-hydroxy derivative of the formula (4) of desmethyl primaquine shows only a slight gametocytocidal activity. Desmethyl primaquine of the formula (5) shown below and 5-hydroxy of the formula (3) and carboxylic acid of the formula (12) metabolites of PQ are all inactive. Of particular interest is the observation that two of the quinolines of the formulae (10) and (11) shown above with unsubstituted —NH2 group on 8-position are directly sporontocidal. [W. Peters et al, ‘The activity of primaquine and its possible metabolites against rodent malaria’ Primaquine: Pharmacokinetics, Metabolism, Toxicity and Activity, pp 93–101 (1984), John Wiley & Sons].
Toxicity of Primaquine and its Metabolites:
Primaquine of the formula (2) shown below itself appears to have little oxidant activity even when incubated with G-6PD deficient erythrocytes [I. M. Fraser et al, ‘Effects of Drugs and Drug Metabolites on Erythrocytes from Normal and Glucose-6-phosphate Dehydrogenase Deficient Individuals’, Annals of the New York Academy of Sciences, 151, 777–94 (1968)], John Wiley & Sons].
Whereas 5-hydroxyprimaquine of thr formula (3) and 5,6-dihydroxy-8-aminoquinoline of the formula (11) cause oxidation of oxyhemoglobin (HbO2) to methemoglobin (Met Hb) and of reduced glutathione (GSH) [K. A. Fletcher et al, ‘The Pharmacokinetics and Biochemical Pharmacology of Primaquine in Rhesus Monkeys and Rats’ in Primaquine: Pharmacokinetics, Metabolism, Toxicity and Activity, pp 49–63 (1984), John Wiley & Sons].
The carboxylic acid of the formula (12), a major metabolite of primaquine circulating in the plasma, has not shown any antimalarial activity. It is uncertain whether it contributes significantly to the toxicity of primaquine although it does not cause methemoglobin formation in vitro. Earlier we reported causal prophylactic activity of primaquine derivative namely N1-(3-acetyl-4,5-dihydro-2-furanyl)-N4-(6-methoxy-8-quinolinyl)-1,4-pentanediamine at 3.16 mg/kg×3 doses against sporozoite induces P. cynomolgi B. infection in monkeys. The derivative also exerts anti-relapse (radical curative) activity at 1 mg/kg×7 days (G. P. Dutta, S. K. Puri, V. C. Pandey, M. Seth, A. P. Bhaduri, S. K. Chatterjee, O. P. Asthana and K. C. Gupta, Tropical Diseases, 286 (1998), G. P. Dutta, S. K. Puri, A. P. Bhaduri and M. Seth, Am. J. Trop. Med. Hyg. 41, 635, (1989). In the derivative, primaquine is substituted at primary amino functionality.
Thus from the above studies, it is obvious that primaquine possesses antimalarial activities such as blood schizontocidal, tissue schizontoidal and gametocytocidal which are also exhibited by its metabolites. Primaquine is even more active than its metabolites. The carboxylic acid of the formula (12) though a major metabolite, is non-functional. The metabolites of primaquine are also responsible for its toxicity. The tricyclic metabolite of the formula (8) is active but less toxic which therefore, suggests the significance of intact side chain. Therefore, if primaquine molecule is manipulated through the side chain possibly toxicity could be modulated. Secondly, primaquine is absorbed and metabolized very fast and as a consequence, oxidative burst accrues very fast. Therefore, its controlled delivery may result in less toxicity. This led us to prepare primaquine prodrug of less toxic profile. Primaquine is of a basic nature with a free amino functionality, which is a point of metabolism for inactive metabolite. We derivatised this amino functionality to enaminone and evaluated its efficacy for gametocytocidal action and methemoglobin toxicity. Enaminones are a functional group for controlled delivery of amino drugs. An enaminone derivative of a physiologically active amine may well show improved transport across biological membranes and allow a high concentration of the amine to be released close to the site of action. This functional group provides resistance towards hydrolytic cleavage at acidic pH as compared to the plain amine. We prepared enaminone derivative of primaquine shown in formula (1) on two accounts. Firstly, it should have slow metabolic degradation through side chain and secondly, compound of enhanced lipophilic character should penetrate better in the tissue, especially in the liver where hypnozoites reside. We therefore, embarked on the preparation of enaminone derivative of formula (1) and the results of its gametocytocidal effects and its safety profiles are mentioned here. As already mentioned earlier at the beginning, the search for a safe gametocytocidal agent is needed for two reasons, firstly, to block the recurrence of malaria in non-endemic regions where malaria has already been eradicated through vector control methods by individuals visiting endemic regions, and secondly, to block spread of even resistant strains.
Primaquine and its putative metabolites are shown below:
